Communication between neurons relies on the release of neurotransmitters from pre-synaptic nerve terminals. Release is triggered by increases in intracellular [Ca2+] and is mediated by the fusion of transmitter-filled synaptic vesicles with the plasma membrane. The molecular mechanism that couples Ca2+ to exocytosis is not known. Synaptotagmin is a Ca2+ binding protein that has been proposed to function as a Ca2+ sensor that triggers release. We propose to examine the function of this protein, primarily on its two putative Ca2+-sensing domains, C2A and C2B. The structure and Ca2+-binding properties of the C2A-domain have been previously studied in detail. However, little is known concerning the function of the C2B-domain. We hypothesize that C2B is a Ca2+-sensing module that plays a critical role in Ca2+-triggered exocytosis. Support for this hypothesis is provided by preliminary data indicating that C2B must bind Ca2+, change conformation and oligomerize in order for docked synaptic vesicles to fuse in response to stimulation in vivo. To determine how C2B functions in exocytosis, three Specific Aims are proposed. (1) A series of biochemical studies will examine the kinetics of Ca2+-C2B interactions, the Ca2+ requirements for synaptotagmin oligomerization, and the role of C2B in the facilitation of SNARE complex assembly. (2) Time resolved amperometry, as well as genetic manipulations of Drosophila, will be used to address the role of C2B in fusion pore dynamics and in excitation-secretion coupling. A critical preliminary finding is that synaptotagmin regulates the opening and dilation kinetics of fusion pores, placing synaptotagmin at the final stages of the fusion reaction that is mediated by SNAREs. This finding supports the hypothesis that synaptotagmins and SNAREs are constituents of Ca2+-regulated exocytotic fusion pores. (3) Biochemical studies will be carried out to determine the subunit stoichiometry of this complex, its morphology, and the interfaces that mediate its assembly. The ability of the release machinery to respond to Ca2+ is subject to modulation and is likely to comprise an important locus for synaptic plasticity. Thus, a better understanding of the release process will provide insights into novel modes of synaptic plasticity and should ultimately provide targets for the treatment of diseases in which synaptic transmission is impaired.